Mouse Preimplantation Embryonic Development Research Articles

P-203 The Effect of c-Abl Tyrosine Kinase Inhibition in Cell Fate Specification and Morphogenesis During Preimplantation Mouse Embryo

Abstract Study question What are the mechanisms governing the regulation of c-Abl tyrosine kinase in preimplantation embryo development? Summary answer Inhibition of c-Abl impairs the fate determination of TE and ICM cells during mouse preimplantation embryo development, preventing cell specilization after compaction. What is known already The successful completion of polarization, compaction and lineage specification stages is required for the embryo to reach the blastocyst stage from the zygote stage during preimplantation embryonic development. c-Abl is a tyrosine kinase localized in cell nucleus and cytoplasm and also capable of nuclear-cytoplasmic shuttling. c-Abl is activated when DNA damage occurs and plays a role in the DNA repairment mechanism. The small-molecule inhibitor imatinib is classified as type II inhibitors which targets the inactive conformation of the kinase domain and specifically inhibit c-Abl. Study design, size, duration C57BL6 female mice at 6-8-wk old were superovulated with 7,5 IU PMSG and after 48 hr 7,5 IU hCG, then mated. 20 hours after hCG enjection, zygotes were collected and cultured for 96h at 5% CO2, 5% O2 at 37oC. We designed 5 experimental groups: control, DMSO, 1 µM, 5 µM and 10 µM (in KSOM+AA) imatinib group. To determine how altering the imatinib might affect embryonic growth and development, morphokinetic parameters were evaluated. Participants/materials, setting, methods Immunofluorescence staining for embryos was performed after 4% paraformaldehyde fixation. Then embryos incubated c-Abl, YAP, TEAD4, PARD6, E-cadherin and CDX2. DAPI mounting medium used for nucleus staining. Imaging was performed under a confocal microscope. Fluorescent intensity analysis was performed with ImageJ. We detected c-Abl, Yap, Tead, Cdx2, Oct4 and Nanog mRNA expression levels using the qRT-PCR. For the statistical analysis we used GraphPad Prism (2-Way ANOVA and and One-way ANOVA (used Geisser-Greenhouse correction)). Main results and the role of chance Treatment of zygotes with imatinib resulted in embryonic developmental arrest (control, 81%; 1 µM imatinib, 58.5%; 5 µM imatinib, 54%, 10 µM imatinib 0%) and led to a significant decrease in the rate of blastocyst formation. We detected that the expression level of c-Abl was decreased in the imatinib groups compared to the control group. We determined that YAP and TEAD, which should be localized in the nuclei of TE cells, lost their nuclear expression patterns and were expressed in the cytoplasm and their expression levels decreased. We detected the PARD6 pattern, which was evident at the cell borders in the control group, distributed throughout the cytoplasm in the imatinib groups. While cytoplasmic and decreased localization of CDX2, which should be nuclear in TE cells, was determined in imatinib groups, we also defined that E-cadherin distribution was impaired at cell borders compared to the control group. c-Abl mRNA level was decreased in the 1 and 5 µm imatinib groups, Yap mRNA level was decreased in the 5 µm imatinib group. Cdx2 and Nanog mRNA levels were decreased in the 1 and 5 µm imatinib group (*p<0.1, **p <0.01, ***p <0.001, ****p <0.0001). Limitations, reasons for caution All animals used in this study were obtained from Yeditepe University Faculty of Medicine Experimental Research Center (YUDETAM) and all the experimental procedures have been approved by Yeditepe University Ethical Committee. Wider implications of the findings Imatinib significantly inhibited the development of eight-cell embryos to the blastocyst stage compared to controls a concentration-dependent manner, furthermore, c-Abl inhibition affected linegae specification by changing the localization of YAP/TEAD, markers of lineage differentiation. Therefore, c-Abl can regulate cell specification and cell fate determination during mouse preimplantation embryonic development. Trial registration number YAP-AP-SAB-21019

O3-4 - Effects of conditioned media from polarized macrophages on mouse pre-implantation embryonic development and outgrowth of blastocysts

O3-4 - Effects of conditioned media from polarized macrophages on mouse pre-implantation embryonic development and outgrowth of blastocysts

Knockdown of Toe1 causes developmental arrest during the morula-to-blastocyst transition in mice

The target of EGR1 protein 1 (TOE1) is evolutionarily conserved from Caenorhabditis elegans to mammals, which plays a critical role in the maturation of a variety of small nuclear RNAs. Mutation in human TOE1 has been reported to cause pontocerebellar hypoplasia type 7, a severe neurodegenerative syndrome. However, the role of TOE1 in early embryonic development remains unclear. Herein, we found that Toe1 mRNA and protein were expressed in mouse preimplantation embryos. Silencing Toe1 by siRNA led to morula-to-blastocyst transition failure. This developmental arrest can be rescued by Toe1 mRNA microinjection. EdU incorporation assay showed a defect in blastomere proliferation within developmentally arrested embryos. Further studies revealed that Toe1 knockdown caused increased signals for γH2AX and micronuclei, indicative of sustained DNA damage. Moreover, mRNA levels of cell cycle inhibitor p21 were significantly upregulated in Toe1 knockdown embryos before developmental arrest. Together, these results suggest that TOE1 is indispensable for mouse early embryo development potentially through maintaining genomic integrity. Our findings provide further insight into the role of TOE1 in mouse preimplantation embryonic development.

Protective Effect of Minocycline Hydrochloride on the Mouse Embryonic Development Against Suboptimal Environment.

Numerous studies have reported how inner cell mass (ICM) and trophectoderm (TE) was determined during the process of early mouse embryonic development from zygotes into organized blastocysts, however, multiple mysteries still remain. It is noteworthy that pluripotent stem cells (PSCs), which are derived from embryos at different developmental stages, have identical developmental potential and molecular characteristics to their counterpart embryos. Advances of PSCs research may provide us a distinctive perspective of deciphering embryonic development mechanism. Minocycline hydrochloride (MiH), a critical component for maintaining medium of novel type of extended pluripotent stem cells, which possesses developmental potential similar to both ICM and TE, can be substituted with genetic disruption of Parp1 in our previous study. Though Parp1-deficient mouse ESCs are more susceptible to differentiate into trophoblast derivatives, what role of MiH plays in mouse preimplantation embryonic development is still a subject of concern. Here, by incubating mouse zygotes in a medium containing MiH till 100 h after fertilization, we found that MiH could slow down embryonic developmental kinetics during cleavage stage without impairing blastocyst formation potential. Olaparib and Talazoparib, two FDA approved PARP1 inhibitors, exhibited similar effects on mouse embryos, indicating the aforementioned effects of MiH were through inhibiting of PARP1. Besides, we showed an embryonic protective role of MiH against suboptimal environment including long term exposure to external environment and H2O2 treatment, which could mimic inevitable manipulation during embryo culture procedures in clinical IVF laboratory. To our knowledge, it is not only for the first time to study MiH in the field of embryo development, but also for the first time to propose MiH as a protective supplement for embryo culture, giving the way to more studies on exploring the multiple molecular mechanisms on embryonic development that might be useful in assisted reproductive technology.

Cell fate determination and Hippo signaling pathway in preimplantation mouse embryo.

First cell fate determination plays crucial roles in cell specification during early phases of embryonic development. Three classical concepts have been proposed to explain the lineage specification mechanism of the preimplantation embryo: inside-outside, pre-patterning, and polarity models. Transcriptional effectors of the Hippo signal pathway are YAP and TAZ activators that can create a shuttle between the cytoplasm and the nucleus. Despite different localizations of YAP in the cell, it determines the fate of ICM and TE. How the decisive cue driving factors that determine YAP localization are coordinated remains a central unanswered question. How can an embryonic cell find its position? The objective of this review is to summarize the molecular and mechanical aspects in cell fate decision during mouse preimplantation embryonic development. The findings will reveal the relationship between cell-cell adhesion, cell polarity, and determination of cell fate during early embryonic development in mice and elucidate the inducing/inhibiting mechanisms that are involved in cell specification following zygotic genome activation and compaction processes. With future studies, new biophysical and chemical cues in the cell fate determination will impart significant spatiotemporal effects on early embryonic development. The achieved knowledge will provide important information to the development of new approaches to be used in infertility treatment and increase the success of pregnancy.

PTHrP promotes development of mouse preimplantation embryos through the AKT/cyclin D1 pathway and nuclear translocation of HDAC4.

Parathyroid hormone-related protein (PTHrP), the main cause of humoral hypercalcemia in malignancies, promotes cell proliferation and delays terminal cell maturation during embryonic development. Our previous study reported that PTHrP plays important roles in blastocyst formation, pluripotency gene expression, and histone acetylation during mouse preimplantation embryonic development. In this study, we further investigated the mechanism of preimplantation embryonic development regulated by PTHrP. Our results showed that Pthrp depletion decreased both the developmental rate of embryos at the cleavage stage and the cell number of morula-stage embryos. Pthrp-depleted embryos had significantly decreased levels of cyclin D1, phospho (p)-AKT (Thr308) and E2F1. However, Pthrp depletion did not cause significant changes in CDK4, β-catenin or RUNX2 expression. In addition, our results indicated that Pthrp depletion promoted HDAC4 translocation from the cytoplasm to the nucleus in cleavage-stage embryos by stimulating the activity of protein phosphatase 2A (PP2A), which resulted in dephosphorylation of HDAC4. Taken together, these results suggest that PTHrP regulates cleavage division progression and blastocyst formation through the AKT/cyclin D1 pathway and that PTHrP modulates histone acetylation patterns through nuclear translocation of HDAC4 via PP2A-dependent HDAC4 dephosphorylation during preimplantation embryonic development in mice.

Synthesis and maintenance of lipid droplets are essential for mouse preimplantation embryonic development.

Lipid droplets (LDs), which are ubiquitous organelles consisting of a neutral lipid core coated with a phospholipid monolayer, play key roles in the regulation of cellular lipid metabolism. Although it is well known that mammalian oocytes and embryos contain LDs and that the amount of LDs varies among animal species, their physiological functions remain unclear. In this study, we have developed a method based on two-step centrifugation for efficient removal of almost all LDs from mouse MII oocytes (delipidation). We found that delipidated MII oocytes could be fertilized in vitro, and developed normally to the blastocyst stage even when the embryos were cultured in the absence of a fatty acid supply. LDs were newly synthesized and accumulated soon after delipidation, but chemical inhibition of long chain acyl-CoA synthetases (ACSLs) blocked this process, resulting in severe impairment of early embryonic development. Furthermore, we found that overabundance of LDs is detrimental to early embryonic development. Our findings demonstrate the importance of synthesis and maintenance of LDs, mediated in part by ACSL activity, during preimplantation embryonic development.

Long non-coding RNAs potentially function synergistically in the cellular reprogramming of SCNT embryos

BackgroundLong non-coding RNAs (lncRNAs), a type of epigenetic regulator, are thought to play important roles in embryonic development in mice, and several developmental defects are associated with epigenetic modification disorders. The most dramatic epigenetic reprogramming event occurs during somatic cell nuclear transfer (SCNT) when the expression profile of a differentiated cell is abolished, and a newly embryo-specific expression profile is established. However, the molecular mechanism underlying somatic reprogramming remains unclear, and the dynamics and functions of lncRNAs in this process have not yet been illustrated, resulting in inefficient reprogramming.ResultsIn this study, 63 single-cell RNA-seq libraries were first generated and sequenced. A total of 7009 mouse polyadenylation lncRNAs (including 5204 novel lncRNAs) were obtained, and a comprehensive analysis of in vivo and SCNT mouse pre-implantation embryo lncRNAs was further performed based on our single-cell RNA sequencing data. Expression profile analysis revealed that lncRNAs were expressed in a developmental stage-specific manner during mouse early-stage embryonic development, whereas a more temporal and spatially specific expression pattern was identified in mouse SCNT embryos with changes in the state of chromatin during somatic cell reprogramming, leading to incomplete zygotic genome activation, oocyte to embryo transition and 2-cell to 4-cell transition. No obvious differences between other stages and mouse NTC or NTM embryos at the same stage were observed. Gene oncology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and weighted gene co-expression network analysis (WGCNA) of lncRNAs and their association with known protein-coding genes suggested that several lncRNAs and their associated with known protein-coding genes might be involved in mouse embryonic development and cell reprogramming.ConclusionsThis is a novel report on the expression landscapes of lncRNAs of mouse NT embryos by scRNA-seq analysis. This study will provide insight into the molecular mechanism underlying the involvement of lncRNAs in mouse pre-implantation embryonic development and epigenetic reprogramming in mammalian species after SCNT-based cloning.

Expression and Potential Role of microRNA-29b in Mouse Early Embryo Development

Background/Aims: MicroRNA-29b (miR29b) has been previously identified in early mouse embryos through miRNA microarray analysis. Recent research has indicated that miR29b participates in DNA methylation by regulating DNA methyltransferase 3a/3b (Dnmt3a/3b) expression. However, the expression pattern and biological function of miR29b in mouse preimplantation embryonic development remain unknown. Methods: In this study, we examined the expression patterns of miR29b and Dnmt3a/3b in mouse early embryos at different developmental stages. Subsequently, expression and localization of DNMT3A/3B protein was analyzed in mouse early embryos by immunofluorescence staining. The biological function of miR29b in mouse early embryos was analyzed by microinjection of commercially available miRNA-specific inhibitors and mimics. Results: Our data showed that Dnmt3a/3b mRNA expression is negatively regulated by miR29b in mouse early embryos. Immunofluorescence analysis revealed that DNMT3A/3B protein expression is predominantly localized within the nucleoplasm of embryos. Alterations to the activity of miR29b could change the DNA methylation levels in mouse preimplantation embryos and lead to a developmental blockade, from the morula to the blastocyst stage. Conclusion: These results indicated a role for the miR29b-Dnmt3a/3b-DNA methylation axis in mouse early embryonic development, and we provide evidence that miR29b is indispensable for mouse early embryonic development. This study contributes to a preliminary understanding of the role of miR29b during mouse embryonic development.

Identification of 4438 novel lincRNAs involved in mouse pre-implantation embryonic development.

Long intergenic non-coding RNAs (lincRNAs) as a key group of non-coding RNAs have gained substantial attention. Though lincRNAs have been systematically explored in various mouse tissues and cell lines, large-scale identification of lincRNAs in mouse pre-implantation embryonic development (PED) process has not be documented previously. Therefore, it is important to identify and characterize novel lincRNAs that may be involved in PED. In this paper, we performed transcriptome assembly based on published single-cell RNA-seq data during mouse PED and identified 4,438 putative lincRNAs. Combining these with Ensembl lincRNAs, we established a reference catalog of 5,808 transcribed lincRNAs in PED. We then systematically analyzed the lincRNAs in this reference catalog and revealed that the identified novel PED lincRNAs are generally comparable with known Ensembl lincRNAs in genomic aspects. In addition, the global expression patterns can be separated by zygote first cleavage division in clustering analysis and we further identified and analyzed differentially expressed lincRNAs involved in this process. The expression of lincRNAs involved in the process is negatively correlated with promoter methylation in trend. The identified lincRNAs involved in zygote first cleavage division could have important roles in mouse early embryonic development and need further functional studies. Altogether, a novel reference catalog of mouse PED lincRNAs is provided and characterized, which would be a valuable resource for further functional analyses and may help elucidate the pre-implantation regulatory mechanism.

Analysis of transcription factor Stk40 expression and function during mouse pre-implantation embryonic development

Determining the molecular mechanisms in the regulation of early embryonic development is crucial for assisted reproductive technology clinical applications. Serine/threonine protein kinase 40 (Stk40) is a member of the serine/threonine kinase family. It is essential in diverse signaling pathways associated with a wide range of cellular activities, including proliferation, differentiation, survival and apoptosis. However, its involvement and molecular mechanisms in pre‑implantation embryonic development have not been well‑defined. In the present study, it was demonstrated that Stk40 was involved in the development of mouse pre‑implantation embryos. Immunofluorescence and confocal microscopy analyses showed that Stk40 was equally expressed in the nuclei and cytoplasm during all stages of pre‑implantation mouse embryos of imprinting control region mice. Reverse transcription‑polymerase chain reaction showed a significantly higher transcription rate of Stk40 mRNA in the two‑cell stage. The results demonstrated that Stk40 downregulation by microinjection of small interfering RNA into the mouse zygote markedly decreased the blastulation compared with that in the control (Stk40i‑1 vs. control: 65.2% and 77.0%, P<0.05 and Stk40i‑2 vs. control: 49.8% and 70.1%, respectively, P<0.05). In addition, silencing of Stk40 significantly increased the transcription rate of reticulocalbin‑2, whereas that of the homeobox protein, Cdx2, was decreased. In conclusion, the results suggested that Stk40 may be critical in the development of pre‑implantation embryos.

The Roles of Parathyroid Hormone-Like Hormone during Mouse Preimplantation Embryonic Development

Parathyroid hormone-like hormone (PTHLH) was first identified as a parathyroid hormone (PTH)-like factor responsible for humoral hypercalcemia in malignancies in the 1980s. Previous studies demonstrated that PTHLH is expressed in multiple tissues and is an important regulator of cellular and organ growth, development, migration, differentiation, and survival. However, there is a lack of data on the expression and function of PTHLH during preimplantation embryonic development. In this study, we investigated the expression characteristics and functions of PTHLH during mouse preimplantation embryonic development. The results show that Pthlh is expressed in mouse oocytes and preimplantation embryos at all developmental stages, with the highest expression at the MII stage of the oocytes and the lowest expression at the blastocyst stage of the preimplantation embryos. The siRNA-mediated depletion of Pthlh at the MII stage oocytes or the 1-cell stage embryos significantly decreased the blastocyst formation rate, while this effect could be corrected by culturing the Pthlh depleted embryos in the medium containing PTHLH protein. Moreover, expression of the pluripotency-related genes Nanog and Pou5f1 was significantly reduced in Pthlh-depleted embryos at the morula stage. Additionally, histone acetylation patterns were altered by Pthlh depletion. These results suggest that PTHLH plays important roles during mouse preimplantation embryonic development.

Effects of Inflammatory Conditions from Coculture with Activated Macrophages on Mouse Preimplantation Embryonic Development In Vitro.

Inflammatory conditions are involved in important reproductive events of mammals such as, ovulation, menstruation, implantation, labor and delivery. The inflammatory milieu in reproductive organs may have various effects on preimplantation embryonic development. The aim of this study was to evaluate the effects of inflammatory conditioning on mouse preimplantation embryonic development in vitro. To provide an inflammatory condition for preimplantation embryos, 2-cell stage embryos were cocultured with the RAW 264.7 cells, a mouse monocyte/macrophage cell line, which were activated by treatments of lipopolysaccharide (LPS) before coculture. The inflammatory response-like condition by LPS was evaluated and confirmed by RT-PCR for inflammation related genes (IL-1α, IL-6, iNOS, Cox-2, MCP-1 and TNF-α) and ELISA for secreted cytokines (IL-1α, IL-6 and MCP-1). Mouse 2-cell stage embryos were collected from oviduct and cultured in the Quinn's Advantage Blastocyst Medium (QABM) supplemented with 10% serum protein substitute (SPS) as control (CON), or directly cocultured with either intact (INT) or LPS activated (ACT) RAW 264.7 cells in the QABM without SPS. At post-hCG 120 h (72 h after culture), the blastulation rate of ACT (98.1%) was higher than that of CON (89.1%) and INT (94.5%) while there was no statistical significance between them. In addition, the hatching rate of ACT (54.5%) was higher than that of CON (47.3%) and INT (36.4%) without statistical significance. However, the mean cell number of blastocysts in ACT (112.0±6.9) was significantly higher (p<0.01) than those of CON (84.9±6.9) and INT (82.5±4.4), whereas there was no significant difference in apoptotic index among them. To examine whether embryotrophic effects of the inflammatory response-like condition on preimplantation embryos in vitro are induced by direct cell-to-cell contact, 2-cell embryos were indirectly cocultured with RAW 264.7 cells using cell culture inserts, which prevent direct contact between embryos and cocultured cells. Blastulation and hatching rates of ACT (94.1% and 47.1%) were slightly higher than that of CON (84.0% and 32.0%) and INT (90.9% and 32.7%) without statistical significance in indirect coculture system. Furthermore, the mean cell number of blastocysts in ACT (106.2±5.7) was also significantly higher (p<0.01) than those of CON (73.8±5.4) and INT (89.3±6.3) with no difference in apoptotic index. Collectively, this study demonstrates that the inflammatory response-like condition induced by activated RAW 264.7 cells could increase cell proliferation of mouse preimplantation embryos in vitro. Our results suggest that a certain inflammatory conditions could improve developmental competence of mouse preimplantation embryos in vitro by providing embryotrophic cytokines and factors. This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0004871). (poster)

Preimplantation development of mouse: A view from cellular behavior

A mature animal body contains a variety of different cell types, and these cells are distributed in a well-organized fashion along the body axes. One of the major questions in developmental biology is how cells acquire different characteristics. In addition, it is important to understand how the embryo forms the body axes and how cells are allocated along these axes during development. Among mammalian species, the molecular mechanisms that regulate embryonic development have been well analyzed and characterized in mice. Here, mouse preimplantation embryonic development is briefly summarized and our current understanding of this complex process based on recent observations is reviewed.

Expression and transient nuclear translocation of proprotein convertase 1 (PC1) during mouse preimplantation embryonic development

Preimplantation embryos express a number of hormones, neuropeptides, and membrane receptors known to derive from proteolytic activation of their precursors by the seven-member family of subtilisin-like, calcium-dependent serine proteinases known as proprotein convertases (PCs). The goal of this study was to determine the pattern of PC expression in mouse preimplantation embryos. Transcripts for all PCs, except PC2, were detected by reverse transcription-polymerase chain reaction (RT-PCR) in unfertilized and fertilized eggs. Furin, PACE4, PC1, and PC7 transcripts remained present at subsequent stages of preimplantation embryonic development, whereas the levels of transcripts for PC4 and PC5 gradually disappeared after the 2-cell stage. Proprotein convertase 1 (PC1) expression was further examined at the protein level. Immunoblotting revealed the presence of the zymogen and mature forms of this enzyme in eggs and embryos. Immunofluorescence laser confocal microscopy showed PC1-specific staining throughout the cytoplasm of unfertilized eggs. After fertilization, surprisingly, the staining was concentrated in pronuclei. It relocated to the cytoplasm at postzygotic stages and was particularly strong at junctions between blastomeres. The nuclear translocation of PC1 in fertilized eggs is probably mediated by its prodomain. Indeed, when transduced in human colon carcinoma LoVo cells, a mutant proPC1 incapable of cleaving off its prodomain was shown to accumulate in the nucleus. Furthermore, when N-terminally fused to green fluorescent protein, this domain was able to direct the reporter protein to the nucleus of these cells. Collectively, these data establish that eggs and preimplantation embryos express various PCs necessary for proteolytic activation of precursors of hormones and growth factors. They also raise the possibility of a nuclear function for PC1 during zygote formation.

DNA topoisomerase II is essential for preimplantation mouse development.

Topoisomerase II (topo II) is an essential enzyme that alters DNA topology. This activity is important for a variety of chromosome functions including DNA replication, transcription, recombination, and chromosome condensation and segregation. Previously we localized topo II in mouse gametes and preimplantation embryos using isoform-specific antibodies demonstrating the presence of the enzyme in oocytes and embryos, but not sperm. To probe functions of topo II during preimplantation development, we treated mouse zygotes with 100 nM teniposide, and assessed embryo morphology and DNA replication. Teniposide blocked cleavage in 69% embryos; the remainder cleaved once but had abnormal nuclei. Teniposide-treated embryos were devoid of topo II immunofluorescence. Teniposide also prevented DNA replication, implicating topo II in this process. Embryos treated with a 2 hr pulse of teniposide recovered and developed to the blastocyst stage, indicating 100 nM teniposide did not induce apoptosis. To more specifically analyze topo IIalpha function, we treated zygotes with topo IIalpha-targeted antisense oligodeoxynucleotides. Most zygotes arrested at the 2-cell stage while controls developed into blastocysts indicating topo IIalpha is essential for preimplantation development. The absence of topo IIalpha, but not beta immunofluorescence in antisense-treated embryos confirms the specificity and impact of the treatment. In addition, topo IIalpha is newly synthesized at the 2-cell stage. These results establish an essential function for topo II in mouse preimplantation embryonic development.

P-158. The effect of different doses of i.v. administration of platelet activating factor on mouse preimplantation embryonic development in vitro

P-158. The effect of different doses of i.v. administration of platelet activating factor on mouse preimplantation embryonic development in vitro

Differential expression of Ped gene candidates in preimplantation mouse embryos.

The Ped (preimplantation embryonic development) gene influences the rate of mouse preimplantation embryonic development and subsequent survival. Four similar tandem genes in the Q region of the major histocompatibility complex-Q6, Q7, Q8, and Q9-were identified as Ped gene candidates. In this study, expression of these genes during preimplantation development was examined and quantitated by reverse transcription-polymerase chain reaction and single nucleotide primer extension assays in order to investigate their contribution to the Ped gene phenotype. The Q7/Q9 gene pair was found to be transcribed in preimplantation mouse embryos, whereas transcription of the Q6/Q8 gene pair was undetectable. Both Q7 and Q9 are expressed in embryos from one Ped fast strain, C57BL/6, while only the Q9 gene is expressed in another Ped fast strain, B6.K2. These results suggest that both the Q7 and Q9 genes can function as the Ped gene in the mouse. Interestingly, the expression pattern of the Q7 and Q9 genes in preimplantation embryos is the same as in splenic lymphocytes. However, the Q6 and Q8 genes are expressed in splenic lymphocytes but not in preimplantation embryos. Treatment of mouse preimplantation embryos with interferon gamma (gamma-IFN) did not induce expression of the Q6/Q8 genes but enhanced expression of the Q7/Q9 genes. The mechanism of this differential transcription pattern is currently under investigation.

Inhibitory effect of human hydrosalpingeal fluid on mouse preimplantation embryonic development is significantly reduced by the addition of lactate.

Implantation and pregnancy rates following in-vitro fertilization-embryo transfer are reduced in the presence of hydrosalpinges, but the basis of the inhibition is unknown. We examined the effect of hydrosalpingeal fluid on preimplantation development of mouse embryos. Embryos cultured in 100% hydrosalpingeal fluid were significantly inhibited developmentally as compared to embryos cultured in 100% defined medium, which served as controls. In contrast, embryos cultured in 50% hydrosalpingeal fluid/50% defined medium reached the blastocyst stage at the same frequency as the controls. When lactate (final concentration 10 mM) was added to 100% hydrosalpingeal fluid, the percentage of cultured embryos that reached the blastocyst stage was significantly increased as compared to 100% hydrosalpingeal fluid, although the percentage was slightly lower than that observed for embryos cultured in the 100% defined medium. A similar but less pronounced effect occurred when pyruvate was added to hydrosalpingeal fluid. These results do not support the concept that a potent embryotoxic agent is commonly present in hydrosalpingeal fluid. Rather, they are consistent with the notion that the inhibitory effect of hydrosalpingeal fluid on embryonic development is due to the absence of essential factors, and that this deficiency can be largely corrected by the addition of energy sources.

Comparative effects of essential and nonessential metals on preimplantation mouse embryo development in vitro

It is well recognized that deficiencies of essential trace elements during early development can result in structural abnormalities and/or embryonic death. Recently, there has been increasing interest in the concept that small excesses of essential metals can also have negative effects on the developing embryo. We hypothesized that, with respect to toxicity, metals with similar physico-chemical properties would act by similar mechanisms to influence the preimplantation embryo. In the current study we investigated the influence of four essential (Cu, Mn, Fe, Zn), and eight nonessential (Cr, Hg, Pb, V, Al, Ag, Cd, As) metals on mouse preimplantation embryonic development. Two cell stage mouse embryos were cultured for 72 h in media containing varying metal concentrations (0.05–200 μM). Embryo cell differentiation and proliferation were respectively assessed by scoring for blastocyst formation and final embryo cell number. Both nonessential and essential metals were embryotoxic at relatively low concentrations. However, in contrast to our expectations, at similar molar concentrations, redox active essential metals were less toxic than non-redox active nonessential metals. These data suggest that direct metal binding to critical membrane sites and/or intracellular ligands, including protein and nucleic acids, may trigger abnormal development and death prior to metal-associated oxidative damage.